Cationic and polycationic amphiphiles, reactives containing the same and their use

ABSTRACT

The invention concerns new cationic or polycationic amphiphiles which are capable of forming aggregates with macromolecules, in particular with DNA or RNA, and it concerns their delivery into prokaryotic or eukaryotic cells. Compounds with spermyl-dioleoyloxypropyl structure have proven to be particularly preferable.

This application is the U.S. national phase of PCT applicationPCT/EP96/02541, filed Jun. 12, 1996, with a claim to the priority ofGerman Application 19521412-9, filed Jun. 14, 1995.

BACKGROUND OF THE INVENTION

The invention concerns new cationic and polycationic amphiphiles whichare capable of forming aggregates with macromolecules, in particularwith DNA or RNA, and it concerns their delivery into prokaryotic oreukaryotic cells.

Amphiphiles (e.g. surfactants, detergents) have for some time played ageneral role in daily life. In contrast only recently has greaterattention been paid to cationic amphiphiles through the pioneer work ofFelgner since they can be used to deliver DNA and RNA into cells andtransfect them in this manner (P. L. Felgner et al., Proc. Natl. Acad.Sci., USA 84, 7413-7417 (1987); P. Hawley-Nelson. WO 94/05624 et al., J.P. Behr, EP 0394111).

The individual reagents used for this are very varied. This is mostlydue to the fact that they have been found empirically. Thus it has notbeen possible to specifically provide a compound for the application inquestion. Moreover the fact that compounds exist which only have aneffect in a mixture with other reagents reveals the whole complexity oftransfection. A review of most of the reagents that have been found upto now is given by Behr (Bioconjugate Chem., 5, 382-389 (1994) and thereferences cited therein). However, in general the majority of thereagents can form liposomes.

The mechanism of transfection by cationic amphiphiles is still littleunderstood. It appears plausible that the liposomes by means of theirpositive charge form a more positively charged complex with the DNA andthis attaches itself to the negatively polarized cell membrane (P. L.Felgner, Nature 337, 387-388 (1989)). However, it is not certain how thepenetration of the cell membrane and transport to the cell nucleus takesplace.

Nevertheless, apart from the transfection efficiency it is possible todefine a series of other requirements for new reagents:

for efficiency it should not be necessary to pretreat the cells such asby permeabilizing the cell membrane with DMSO, a detergent such asdigitonin or by scraping.

the reagents should not be toxic if possible especially not at the mosteffective concentration and should be preferably biologically degradable

it should be possible to use them equally for all cells that come intoconsideration

they should not have a specificity for certain DNA molecules

it should also be possible to use them in vivo. This means that inaddition to toxicity, compatibility with serum is also of majorsignificance. This has often been the reason for a drastic decrease inthe transfection efficiency.

Hence the object of the invention was to provide appropriate reagentswhich equally fulfill these requirements.

SUMMARY OF THE INVENTION

This object is achieved by certain cationic, lipidic amphiphiles thatcan be used to deliver anionic compounds such as e.g. DNA into cells andin this process surprisingly exhibit improved properties with respect tothe aforementioned requirements.

In particular compounds of the general formula I serve as amphiphiles##STR1## in which R₁ represents a saturated or unsaturated C(O)--C₁₋₂₃or saturated or unsaturated C₁₋₂₄ group

A₁ represents an O--R₂ group in which R₂ has the meaning stated for R₁and can be the same as or different from R₁

A₂ represents an NR₃ X or an N⁺ R₃ R₄ R₅ Y⁻ residue in which

R₃, R₄ which are the same as one another or different, representhydrogen, an alkyl group with 1 to 4 C atoms, a (CH₂)_(n) --OH or a(CH₂)_(n) --NH₂ group where n=2-6,

R₅ which can be the same as or different from R₃ or R₄, denoteshydrogen, an alkyl group with 1 to 4 C atoms, a (CH₂)_(n) --OH, a(CH₂)_(n) -halogenide or a ((CH₂)_(m) NH)_(o) --(CH₂)_(n) --NH₂ group inwhich m is an integer from 2 to 6 and can be equal to or different fromn or o wherein n can be a number from 2 to 6 and o an integer from 0 to4,

X can, in addition to the meaning for R₅, have the following meaning: anamidically bound amino acid, an amidically bound peptide or polypeptide,a C(O)--CHR₆ N(R₇)₂, a C(O)CHR₆ N⁺ (R₇)₃, a C(O)--CHR₆ N⁺ (R₇)₂ R₈ or aC(O)--CHR₆ NR₇ R₈ group in which

R₆ can be a (CH₂)_(m) --NR₇ R₈, a (CH₂)_(m) --N⁺ (R₇)₂ R₈ or a (CH₂)_(m)--N⁺ (R₇)₃ residue

and m can be a number from 1 to 5,

R₇ represents hydrogen or an alkyl group with 1 to 4 C atoms,

R₈ represents a (CH₂)_(n) --N(R₇)₂ or (CH₂)_(n) --N⁺ (R₇)₃ group inwhich n is a number from 2 to 4 and R₇ can have the meaning stated aboveand

Y is a pharmaceutically acceptable anion,

B₁ is an NH[C(O)--(CH₂)_(p) --NH]_(q) --Z residue in which p is a numberfrom 1 to 6 and q is a number from 0 to 2,

Z represents an amidically bound amino acid, an amidically bound peptideor polypeptide, a C(O)--CHR₆ N(R₇)₂, a C(O)--CHR₆ N⁺ (R₇)₃, a C(O)--CHR₆N⁺ (R₇)₂ R₈ or a C(O)--CHR₆ NR₇ R₈ group and R₆ to R₈ and m have theaforementioned meanings and

B₂ can have the meaning stated for A₁ and the meaning for A₁ is onlyvalid with B₁ and that of A₂ is only valid with B₂.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 and 2 show the transfection efficiencies of selected compounds.

FIGS. 3 to 7 show appropriate reaction schemes for the synthesis ofcompounds according to the invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Those compounds are preferred in which the residues R₁ and A₁ have alkylresidues with 10 to 20 C atoms. In addition those compounds in which A₂has the meaning NR₃ X are preferred and in particular those in which R₃denotes hydrogen and X denotes an amidically bound amino acid, asuitable amino acid derivative, a peptide or polypeptide.

Suitable pharmaceutically acceptable anions Y are in particularhalogenides, monomethylsulfate, acetate, trifluoroacetate and phosphate.

The following compounds according to the invention have proven to beparticularly suitable: 2-(6-carboxyspermyl)-1,3-dioleoyloxy-propylamide,2-(N,N,N,N',N',N'-hexamethylornithyl)-1,3-dioleoyloxy-propylamide,1-(6-carboxyspermyl)-2,3-dioleoyloxy-propylamide and/or2,3-dioleoyloxy-N-(N-(spermyl)-glycyl)-aminopropane as well ascorresponding derivatives thereof. However,2-(6-carboxyspermyl)-1,3-dimyristoyloxy-propylamide,2-(1,1,1,5,5,10,10,14,14,14-decamethyl-6-carboxyspermyl)-1,3-dioleoyloxy-propylamideand/or 2-(N,N,N,N',N',N'-hexamethyllysyl)-1,3-dioleoyloxy-propylamidehave also proven to be suitable

Similar compounds such as for example amine and amidine derivatives ofglycerol and propanediols such as e.g. 2-aminopropane compounds havealready been described for other uses, a suitability for biologicalapplications has however, not been suggested (DE-A-2835369; Woltrom etal., Journal of the Am.Soc. (1951) 73(8), p. 3553; Chem. Abs., 59(8),1963, column 8586, FIG. 6).

Furthermore the present application also concerns the production ofreagents with a transfection property. In this connection the newreagents can be used equally as a solution in a water-miscible solventor as an aqueous liposomal formulation. When evaluating theirtransfection efficiency it was noted that:

in serum-free medium they already usually exhibit a better efficiencythan other commercially available products,

in serum-free medium they surprisingly allow a broader plateau of theDNA/reagent ratio with a similar transfection efficiency,

they surprisingly had the same or a better efficiency in mediumcontaining serum,

the efficiency can be increased further by a liposomal formulation,

surprisingly the compounds proved to be much less toxic than compoundswith a similar transfection efficiency in a serum-free system.

The transfection efficiencies of some selected compounds are shown inFIGS. 1 and 2 in comparison to known reagents.

The compounds according to the invention are synthesized by standardmethods for a person skilled in the art (J. March: Advanced OrganicChemistry; John Wiley & Sons, 1985 and M. Bodanszky; Principles ofPeptide Synthesis; Springer Verlag, 1984) and the end-products areoptionally purified by chromatography in particular by ion exchangerchromatography. Appropriate reaction schemes are shown in FIGS. 3 to 7.

A prerequisite for the production of new transfection reagents was thatthe respective compounds are cationic and are suitable for aggregateformation since up to now it is above all such charged reagents thathave resulted in a transfection (there are examples in which the DNA canbe transported into the cell by means of uncharged reagents. However,for this they have to then be enclosed in liposomes during thepreparation of which the molecule to be transported (e.g. DNA) may bedamaged. In general the efficiency achieved by such complexes has notbeen very high (cf. e.g. A. Cudd, C. Nicolau in Liposome Technology(Gregoriadis Ed.), (1984) CRC Press Inc. Boca Raton, Fla.; S. F. Alino,M. Bobadilla, M. Garcia-Sanz, M. Lejarreta, F. Unda, E. Hilario inBiochem. Biophys. Res. Commun. 192, 174 (1993)).

A further starting point was that the compounds should be able to formliposomes. It is not clear why almost exclusively liposome-formingcompounds are suitable for transfection. The latest findings (H.Gershon, R. Ghirlando, S. B. Guttman, A. Minsky in Biochemistry 32, 7143(1993) and B. Sternberg, F. L. Sorgi, L. Huang in FEBS Letters 356, 361(1994) show that they facilitate the formation of so-called meatballspaghetti structures which are required for transfection. It has stillnot been clarified whether the same applies to all reagents and in whichform this contributes to passage through the cell membrane.

The following de novo syntheses resulted from these considerations whichare shown in the diagrams of FIGS. 3 to 7.

In general several methods are available according to the state of theart for providing appropriate reagents such as for examplepharmaceutical formulations. Liposomal and ethanolic formulations areused in particular whereby the efficiency of the various types ofliposomes can already differ (for the preparation of liposomes see e.g.H. Schreier in "Pharmazie in unserer Zeit" 11, 97 (1982)).

However, the compounds according to the invention cannot only be usedfor transfection as liposomes but also in the form of other aggregates.In the case of liposome as well as in the case of aggregate formationother lipidic compounds can additionally be present apart from thecompounds according to the invention. For example compounds from thephospholipid class are suitable for this; other compounds well-known toa person skilled in the art could also be used.

It must also be regarded as surprising that liposomes can be formed fromthe compounds according to the invention of type 3, 6, 12 or 15 (seeexamples 3, 6, 12 and 15) which are described here for the first time.

The transfection was carried out on HeLa cells with the pSV2-CAT plasmidas an example (Gorman, C. M. et al., Mol. Cell. Biol. 2, 1044-1051(1982)). The transfection efficiency is determined by the CAT-ELISA kit(Boehringer Mannheim GmbH) and is shown in FIGS. 1 and 2 in comparisonto known reagents.

Due to their low toxicity these compounds (alone or in combination withother lipidic compounds) can also be used in vivo.

The invention is elucidated further by the following examples.

EXAMPLE 1 Synthesis of2-(N-tert.-butyloxycarbonyl-L-alanyl)-1,3-dihydroxy propylamide 1

756 mg (4 mmol) Boc-L-alanine and 600 μl (4.3 mmol) triethylaminedissolved in 10 ml absolute THF were placed in a round-bottom flask witha bubble counter and cooled to -10° C. Then 320 μl (4 mmol) methylchloroformate was added and it was stirred for 30 minutes at 0° C. Afteraddition of 456 mg (5 mmol) 2-amino-1,3-propanediol sufficient water wasadded to form a homogeneous solution and it was stirred for a further 1h at room temperature. After removing the THF 50 ml ethyl acetate wasadded. The organic phase was washed with saturated NaHCO₃ solution andsaturated NaCl solution and dried over Na₂ SO₄. After removing thesolvent 332 mg (32%) of a colourless oil was obtained.

¹ H-NMR (CDCl₃): δ=1.32 (d; 3 H; J=7.1 Hz; CH--CH₃); 1.40 (s; 9 H;C(CH₃)₃); 3.55-3.85 (m; 4 H; CH(CH₂ OH)₂); 3.85-4.0 m; 1 H; NH--CH);4.0-4.5 (m; 3 H; NH--CH, 2×OH); 5.6-5.8 (m; 1 H; NH--CH(CH₂ OH)₂);7.0-7.35 (m; 1 H; OOC--NH--CH).

¹³ C-NMR (CDCl₃): δ=18.4 (CH--CH₃); 28.2 (C(CH₃)₃), 52.2 (NH--CH--CO);52.4 (CH(CH₂ OH)₂); 61.8 (CH(CH₂ OH)₂); 80.2 (C(CH₃); 155.7 (NH--COO);173.8 (CH--CO--NH).

EXAMPLE 2 Synthesis of2-(N-tert.-butyloxycarbonyl-L-alanyl)-1,3-dioleoyloxy propylamide 2

A solution of 598 mg (2.28 mmol) 1, 1.610 g (5.70 mmol) oleic acid,1.176 g (5.70 mmol) DCC and 28 mg (0.23 mmol) dimethylaminopyridine in40 ml abs. CH₂ Cl₂ was stirred overnight at room temperature in around-bottom flask. Subsequently it was filtered and the filtrate wasrotary evaporated. The crude product was purified by columnchromatography (silica gel, CH₂ Cl₂ :MeOH=30:1). 1.450 g (80%) of acolourless oil was obtained.

¹ H-NMR (CDCl₃): δ=0.87 (t; 6 H; J=6.6 Hz; 2×CH₂ --CH₃); 1.15-1.5 (m; 43H; CH--CH₃, 2×CO--(CH₂)₂ --(CH₂)₄, 2×(CH₂)₆ --CH₃); 1.43 (s; 9 H;C(CH₃)₃); 1.5-1.7 (m; 4 H; 2×CO--CH₂ --CH₂); 1.9-2.1 (m; 8 H; 2×CH₂--CH═CH--CH₂); 2.30 (t, 4 H; J=7.6 Hz; 2×CO--CH₂); 4.0-4.25 (m; 5 H;NH--CH; 2×CH₂ OOC); 4.35-4.5 (m; 1 H; NH--CH); 4.9-5.0 (m; 1 H;NH--CH--CH₂); 5.25-5.45 (m; 4 H; 2×CH═CH); 6.5-6.65 (m; 1 H;OOC--NH--CH).

¹³ C-NMR (CDCl₃): δ=14.0 (2×C(18)H₃); 22.5 (2×C(17)H₂); 24.7 (2×C(3)H₂);27.0, 27.1, 28.98, 29.04, 29.2, 29.4, 29.56, 29.62 {2×[C(4)H₂ --C(7)H₂,C(12)H₂ --C(16)H₂ ]}; 28.1 (C(CH₃)₃); 31.8 (2×C(2)H₂); 33.87, 33.88{2×[C(8)H₂, C(11)H₂ ]}; 47.2 (CH(CH₂ O₂ C--R)₂); 62.4 (CH(CH₂ O₂C--R)₂); 129.6, 129.9 (2×CH═CH); 172.4 (CH--CO--NH); 173.4 (CH₂ --COO).

EXAMPLE 3 Synthesis of 2-L-alanyl-1,3-dioleoyloxy-propylamide 3

702 mg (0.89 mmol) 2 dissolved in 6 ml abs. CH₂ Cl₂ was placed in around-bottom flask with a bubble counter and 2 ml (26.12 mmol) TFA wasadded. After 30 min stirring at room temperature the reaction mixturewas transferred to a separating funnel and 50 ml CH₂ Cl₂ as well as 28ml 1 N NaOH was added. The organic phase was washed with 10 ml saturatedNaHCO₃ solution and dried over Na₂ SO₄. After removing the solvent 599mg (97%) of a nearly colourless oil was obtained.

¹ H-NMR (CDCl₃): δ=0.75-0.9 (m; 6 H; 2×CH₂ --CH₃); 1.1-1.4 (m; 43 H;CH--CH₃, 2×CO--(CH₂)₂ --(CH₂)₄, 2×(CH₂)₆ --CH₃); 1.4-1.7 (m; 6 H; NH₂,2×CO--CH₂ --CH₂); 1.85-2.1 (m; 8 H; 2×CH₂ --CH═CH--CH₂); 2.30 (t, 4 H;J=7.5 Hz; 2×CO--CH₂); 3.35-3.55 (m; 1 H; NH--CH); 4.0-4.25 (m; 4 H;2×CH₂ OOC); 4.3-4.5 (m; 1 H; NH--CH); 5.2-5.4 (m; 4 H; 2×CHH═CH);7.5-7.65 (m; 1 H; CO--NH--CH).

¹³ C-NMR (CDCl₃): δ=14.0 (2×C(18)H₃); 22.5 (2×C(17)H₂); 24.7 (2×C(3)H₂);27.0, 27.1, 28.9, 29.0, 29.2, 29.4, 29.5, 29.6 {2×[C(4)H₂ --C(7)H₂,C(12)H₂ --C(16)H₂ ]}; 31.7 (2×C(2)H₂); 33.9 {2×[C(8)H₂, C(11)H₂ ]}; 46.7(CH(CH₂ O₂ C--R)₂); 62.5 (CH(CH₂ O₂ C--R)₂); 129.5, 129.8 (2×CH═CH);173.3 (CH--CO--NH); 173.4 (CH₂ --COO).

EXAMPLE 4 Synthesis of2-(N,N'-di-tert.-butyloxycarbonyl-L-ornithyl)-1,3-dihydroxypropylamide 4

2.000 g (6 mmol) Boc-L-ornithine and 900 μl (6.45 mmol) triethylaminedissolved in 25 ml absolute THF were placed in a round-bottom flask witha bubble counter and cooled to -10° C. Then 480 μl (6 mmol) methylchloroformate was added and it was stirred for 30 minutes at 0° C. Afteraddition of 684 mg (7.5 mmol) 2-amino-1,3-propanediol sufficient waterwas added to form a homogeneous solution and it was stirred for afurther 1 h at room temperature. After removing the solvent it was takenup in ethyl acetate, washed with saturated NaHCO₃ solution and saturatedNaCl solution and dried over Na₂ SO₄. After removing the solvent 1.987 g(82%) 4 was obtained as colourless crystals.

¹ H-NMR (CDCl₃): δ=1.42 (s; 9 H; C(CH₃)₃); 1.4-1.95 (m; 4 H; CH₂ --CH₂--CH₂ --NH); 3.0-3.2 (m; 2 H; CH₂ NH); 3.6-3.8 (m; 4 H; CH(CH₂ OH)₂);3.9-4.05 (m; 1 H; NH--CH); 4.05-4.6 (m; 3 H; NH--CH, 2×OH); 5.2-5.4 (m;1 H; NH--CH); 5.8-6.05 (m; 1 H; NH--CH); 7.35-7.5 (m; 1 H; NH--CH).

¹³ C-NMR (CDCl₃): δ=26.1 (CH₂ --CH₂ --CH₂ --NH); 28.4 (C(CH₃)₃, 28.5(C(CH₃)₃); 30.1 (CH₂ --CH₂ --NH); 40.0 (CH₂ --NH); 52.9 (CH(CH₂ OH)₂);54.4 (NH--CH--CO); 61.8 (CH(CH₂ OH)₂); 79.3 (C(CH₃)₃); 80.1 (C(CH₃)₃);156.1 (NH--COO); 156.5 (NH-COO); 173.3 (CH--CO--NH).

EXAMPLE 5 Synthesis of2-(N,N'-di-tert.-butyloxycarbonyl-L-ornithyl)-1,3-dioleoxy-propylamide 5

A solution of 1.898 g (4.68 mmol) 4, 3.305 g (11.7 mmol) oleic acid,2.414 g (11.7 mmol) DCC and 57 mg (0.47 mmol) dimethylaminopyridine in50 ml abs. CH₂ Cl₂ was stirred overnight at room temperature in around-bottom flask. Subsequently it was filtered and the filtrate wasrotary evaporated. The crude product was purified by columnchromatography on silica gel (CH₂ Cl₂ :MeOH=30:1). 1.678 g (38%) of theproduct was obtained as a colourless wax-like solid of melting point52-54° C.

¹ H-NMR (CDCl₃): δ=0.75-0.9 (m; 6 H; 2×CH₂ --CH₃); 1.05-1.5 (m; 40 H;2×CO--(CH₂)₂ --(CH₂)₄, 2×(CH₂)₆ --CH₃); 1.42 (s; 18 H; 2×C(CH₃)₃ ;1.45-1.85 (m; 8 H; 2×CH₂ --CH₂ --CH₂ --NH, 2×CO--CH₂ --CH₂); 1.85-2.1(m, 8 H; 2×CH₂ --CH═CH--CH₂); 2.2-2.35 (m; 4 H; 2×CO--CH₂); 3.0-3.35 (2m; 2 H; CH₂ --NH); 4.0-4.25 (m; 5 H; NH--CH; 2×CH₂ OOC); 4.35-4.5 (m; 1H; NH--CH); 4.7-4.8 (m; 1H; NH--CH); 5.1-5.2 (m; 1 H; NH--CH); 5.2-5.4(m; 4 H; 2×CH═CH); 6.65-6.8 (m; 1 H; OOC--NH--CH).

¹³ C-NMR (CDCl₃): δ=14.0 (2×C(18)H₃); 22.5 (2×C(17)H₂); 24.6 (2×C(3)H₂);26.2 (CH₂ --CH₂ --CH₂ --NH); 27.0, 27.1, 28.97, 29.04, 29.2, 29.4,29.56, 29.61 {2×[C(4)H₂ --C(7)H₂, C(12)H₂ --C(16)H₂ ] and CH₂ --CH₂--NH}; 28.1, 28.3 (2×C(CH₃)₃); 31.7 (2×C(2)H₂); 33.85, 33.86 {2×[C(8)H₂,C(11)H₂ ]}; 47.2 (CH(CH₂ O₂ C--R)₂); 62.3 (CH(CH₂ O₂ C--R)₂); 129.6,129.8 (2×CH═CH); 156.3 (NH--COO); 173.30, 173.33 (CH--CO--NH, 2×CH₂--COO).

EXAMPLE 6 Synthesis of 2-L-ornithyl-1,3-dioleoyloxy-propylamide 6

421 mg (0.451 mmol) 5 dissolved in 3 ml abs. CH₂ Cl₂ was placed in around-bottom flask and 1 ml (13.06 mmol) TFA was added. After stirringfor 30 min. at room temperature the reaction mixture was diluted with200 ml CH₂ Cl₂, washed twice with saturated NaHCO₃ solution and driedover Na₂ SO₄. After removing the solvent, 296 mg (89%) of an almostcolourless oil was obtained.

¹ H-NMR (CDCl₃): δ=0.86 (t; 6 H; J=6.7 Hz; 2×CH₂ --CH₃); 1.1-1.45 (m; 40H; 2×CO--(CH₂)₂ --(CH₂)₄, 2×(CH₂)₆ --CH₃); 1.45-1.7 (m; 8 H; CH₂ --CH₂--CH₂ --NH₂, 2×CO--(CH₂ --CH₂); 1.9-2.1 (m; 8 H; 2×CH₂ --CH═CH--CH₂);2.30 (t; 4 H; J=7.5 Hz; 2×CO--CH₂); 2.6-3.1 (m; 4 H; 2×NH₂); 3.15-3.55(m; 2 H; CH₂ --NH₂); 3.55-3.75 (m; 1 H; NH--CH); 4.0-4.25 (m; 4 H; 2×CH₂--OOC); 4.3-4.5 (m; 1 H; NH--CH); 5.2-5.4 (m; 4 H; 2×CH═CH); 7.65-7.75(m; 1 H; CO--NH--CH).

¹³ C-NNR (CDCl₃): δ=14.0 (2×C(18)H₃); 22.5 (2×C(17)H₂); 24.7 (2×C(3)H₂);26.99, 27.04, 29.1, 29.3, 29.5, 29.6, {2×[C(4)H₂ --C(7)H₂, C(12)H₂--C(16)H₂ ], CH₂ --CH₂ --CH₂ NH}; 31.7, (2×C(2)H₂); 33.9 (2×[C(8)H₂,C(11)H₂ ]); 46.7 (CH(CH₂ O₂ C--R)₂); 54.6 (NH₂ --CH--CO); 62.5 (CH(CH₂O₂ C--R)₂); 129.5, 129.8 (2×CH═CH); 173.31, 173.6 (CH--CO--NH, 2×CH₂--COO); 174.7 (CH--CO--NH).

EXAMPLE 7 Synthesis ofN-tert.-butyloxycarbonyl-1,3-dihydroxy-propylamine 7

A solution of 1.07 (5 mmol) Boc₂ O, 697 μl (5 mmol) triethylamine and456 mg (5 mmol) 2-amino-1,3-propanediol in 10 ml THF/water (1:1) wasstirred overnight at room temperature in a round-bottom flask. Afterremoving the THF on a rotary evaporator, 50 ml ethyl aceatate and 10 mlsaturated NaCl solution was added. The organic phase was washed with 10ml saturated NaCl solution and dried over Na₂ SO₄. After removing thesolvent 589 mg (62%) 7 was obtained as colourless crystals. Meltingpoint 83-85° C.

¹ H-NMR (CDCl₃): δ=1.44 (s; 9 H; C(CH₃)₃); 3.4-3.85 (m; 7 H; CH(CH₂OH)₂); 5.3-5.45 (m; 1 H; NC--CH).

¹³ C-NMR (CDCl₃): δ=28.4 (C(CH₃)₃); 53.3 (CH(CH₂ OH)₂); 62.6 (CH(CH₂OH)₂); 79.9 (C(CH₃)₃); 156.5 (NH--COO).

EXAMPLE 8 Synthesis ofN-tert.-butyloxycarbonyl-1,3-dioleoyloxy-propylamine 8

A solution of 478 mg (2.5 mmol) 7, 1.695 g (6 mmol) oleic acid, 1.28 g(6 mmol) DCC and 12 mg (0.1 mmol) dimethylaminopyridine in 25 ml abs.CH₂ Cl₂ was stirred overnight at room temperature in a round-bottomflask. Subsequently it was filtered and the filtrate was rotaryevaporated. The crude product was purified by column chromatography onsilica gel (hexane:ethyl acetate=5:1). 1.482 g (82%) of the product wasobtained as a colourless oil.

¹ H-NMR (CDCl₃): δ=0.86 (t; 6 H; J=6.6 Hz; 2×CH₂ --CH₃); 1.1-1.5 (m; 40H; 2×CO--(CH₂)₂ --(CH₂)₄, 2×(CH₂)₆ --CH₃);.1.43 (s; 9 H; C(CH₃); 1.5-1.7(m; 4 H; 2×CO--CH₂ --CH₂); 1.9-2.1 (m, 8 H; 2×CH₂ --CH═CH--CH₂); 2.30(t; 4 H; J=7.5 Hz; 2×CO--CH₂); 3.95-4.25 (m; 5 H; NH--CH; 2×CH₂ OOC);4.7-4.85 (m; 1 H; OOC--NH--CH); 5.25-5.45 (m; 4 H; 2×CH═CH).

¹³ C-NMR (CDCl₃): δ=14.0 (2×C(18)H₃); 22.5 (2×C(17)H₂); 24.7 (2×C(3)H₂);27.0, 27.1, 28.96, 29.02, 29.2, 29.4, 29.55, 29.62 {2×[C(4)H₂ --C(7)H₂,C(12)H₂ --C(16)H₂ ]}; 28.2 (C(CH₃)₃); 31.8 (2×C(2)H₂); 33.9 {2×[C(8)H₂,C(11)H₂ ]}; 48.4 (CH(CH₂ O₂ C--R)₂); 62.8 (CH(CH₂ O₂ C--R)₂); 79.8(C(CH₃)₃); 129.6, 129.8 (2×CH═CH); 154.9 (NH--COO); 173.3 (CH₂ --COO).

EXAMPLE 9 Synthesis of 1,3-dioleoyloxy-propylamine 9

720 mg (1 mmol) 8, dissolved in 3 ml abs. CH₂ Cl₂ was placed in around-bottom flask and 1 ml (13.06 mmol) TFA was added. After stirringfor 30 min. at room temperature the reaction mixture was diluted with 50ml CH₂ Cl₂, washed with saturated NaHCO₃ solution and dried over Na₂SO₄. After removing the solvent 589 mg (95%) of a colourless oil wasobtained.

¹ H-NMR (CDCl₃): δ=0.87 (t; 6 H; J=6.6 Hz; 2×CH₂ --CH₃); 1.1-1.5 (m; 40H; 2×CO--(CH₂)₂ --(CH₂)₄, 2×(CH₂)₆ --CH₃); 1.35-1.5 (m; 2 H; CH--NH₂);1.5-1.7 (m; 4 H; 2×CO--(CH₂ --CH₂); 1.85-2.1 (m; 8 H; 2×CH₂--CH═CH--CH₂); 2.32 (t; 4 H; J=7.5 Hz; 2×CO--CH₂); 3.2-3.35 (m; 1 H;CH--NH₂); 3.95-4.15 (m; 4 H; 2×CH₂ --OOC); 5.25-5.45 (m; 4 H; 2×CH═CH).

¹³ C-NMR (CDCl₃): δ=14.0 (2×C(18)H₃); 22.5 (2×C(17)H₂); 24.8 (2×C(3)H₂);27.0, 27.1, 28.96, 28.99, 29.03, 29.2, 29.4, 29.55, 29.63 {2×[C(4)H₂--C(7)H₂, C(12)H₂ --C(16)H₂ ]}; 31.8, (2×C(2)H₂); 34.0 (2×[C(8)H₂,C(11)H₂ ]); 49 {CH(CH₂ O₂ C--R)₂ }; 65.7 {CH(CH₂ O₂ C--R)₂ }; 129.6,129.9 (2×CH═CH); 173.4 (CH₂ --COO).

EXAMPLE 10 Synthesis of2-(N,N',N",N'"-tetra-tert.-butyloxy-carbonyl-6-carboxy-spermyl)-1,3-dihydroxy-propylamide10

25 μl (312 μmol) methyl chloroformate was added at -10° C. to a solutionof 202 mg (312 μmol) tetra-Boc-spermine and 47 μl (335 μmol)triethylamine in 2 ml abs. THF and stirred for 30 min at 0° C.

After subsequent addition of 36 mg (390 μmol) 2-amino-1,3-propanediolsufficient water was added to form a homogeneous emulsion and afterwardsit was stirred for a further 1 h at room temperature. After removing thesovent on a rotary evaporator ca. 30 ml ethyl acetate was added. Theorganic phase was washed with saturated NaHCO₃ solution and saturatedNaCl solution and dried over Na₂ SO₄. After removing the solvent 208 mg(93%) of a colourless, foamy solid was obtained.

¹ H-NMR (CDCl₃): δ=1.3-1.47 (m; 36 H; 4×C(CH₃)₃); 1.5-2.05 (m; 8 H;2×N--CH₂ --CH₂ --CH₂ --N, N--CH--CH₂ --CH₂ --CH₂ --N); 2.85-3.4 (m; 10H; 5×N--CH₂); 3.65-3.8 (m; 4 H; 2×CH₂ --OH); 3.8-4.5 (m; 4 H; 2×--CH--,2×CH₂ --OH); 4.8-5.1 (s(broad); 1 H; CH₂ --NH); 5.25-5.4 (s(broad); 1 H;CH₂ --NH); 7.05-7.15 (s(broad); 1 H; NH--CO).

¹³ C-NMR (CDCl₃): δ=25.0, 29.5 (N--CH--CH₂ --CH₂ --CH₂ --N); 28.0, 28.2,28.3 (4×C(CH₃)₃); 38.0, 38.1 (2×N--CH₂ --CH₂ --CH₂ --N); 42.9, 45.0,47.0 (other spermyl-CH₂ carbons); 52.4 (CH(CH₂ --OH)₂); 59.8 (CH--CO);63.1 (CH(CH₂ --OH)₂); 79.3, 79.5, 81.1 (4×CO--O--C)CH₃)₃); 156.0, 156.1(4×CO--O--C)CH₃)₃); 171.1 (CO--NH).

EXAMPLE 11 Synthesis of2-(N,N',N",N'"-tetra-tert.-butyloxycarbonyl-6-carboxy-spermyl)-1,3-dioleoyloxy-propylamide11

A solution of 208 mg (72 μmol) 10, 204 mg (289 μmol) oleic acid, 149 mg(723 μmol) DCC and 4 mg (29 μmol) dimethylamino-pyridine in 6 ml abs.CH₂ Cl₂ was stirred overnight at room temperature in a closedround-bottom flask.

Subsequently it was filtered, rewashed with hexane/ether (3:1) and thefiltrate was rotary evaporated. After column chromatography on silicagel (CH₂ Cl₂ /MeOH=20:1). 160 mg (44%) of a colourless oil was obtained.

¹ H-NMR (CDCl₃): δ=0.8-0.95 (m; 6 H; 2×CH₂ --CH₃); 1.2-1.4 (m; 40 H;2×CO--(CH₂)₂ --(CH₂)₄, 2×(CH₂)₆ --CH₃); 1.4-1.55 (m; 36 H; 4×C(CH₃)₃);1.55-1.75 (m; 10 H; 3×(--CH₂ --CH₂ --CH₂ --)_(sperm), 2×CO--CH₂ --CH₂--); 1.85-2.05 (m, 10 H; (--CH--CH₂ --CH₂ --)_(sperm), 2×CH₂--CH═CH--CH₂); 2.25-2.35 (m; 4 H; 2×CO--CH₂); 3.0-3.35 (m, 10 H;5×N--CH₂); 4.0-4.4 (m; 6 H; 2×CH--N, (CH(CH₂ O₂ C--R)₂); 4.5-5.1 (s(verybroad); 1 H; NH--CH₂); 5.25-5.35 (m; 4 H; 2×CH═CH); 6.8-7.0 (s(broad); 1H; NH--CO).

¹³ C-NMR (CDCl₃): δ=14.4 (2×C(18)H₃); 23.0 (2×C(17)H₂); 25.2 (2×C(3)H₂);27.6 (C(8)H₂,C(11)H₂); 29.5, 29.6, 29.7, 29.9, 30.1, 32.3 ((CH--CH₂--CH₂ --CH₂ --)_(sperm), 2×N--CH₂ --CH₂ --CH₂ --N, 2×C(4)H₂ --C(7)H₂,2×C(12)H₂ --C(16)H₂ ; 28.7, 28.8 (4×C(CH₃)₃); 34.0 (2×C(2)H₂); 38.5,44.6, 46.8, 47.6 (2×N--CH₂ --CH₂ --CH₂ --N, (CH--CH₂ --CH₂ --CH₂--)_(sperm), (CH(CH₂ O₂ C--R)₂); 59.5 (CO--CH); 62.8 (CH(CH₂ O₂ C--R)₂);79.5, 80.1, 81.5 (4×CO--O--C(CH₃)₃); 130.07, 130.37 (2×CH═CH); 156.3(4×CO--O--C(CH₃)₃); 173.7 (CO--NH, 2×CH₂ --COO).

EXAMPLE 12 Synthesis of2-(6-carboxy-spermyl)-1,3-dioleoyloxy-propylamide 12

85 mg (68 μmol) 11 was dissolved in 1 ml abs. CH₂ Cl₂ in a round-bottomflask with bubble counter and 400 μl TFA was added. The reaction mixturewas stirred for 45 min. at room temperature. Subsequently ca. 20 ml CH₂Cl₂ was added, it was washed with saturated NaHCO₃ solution and driedover Na₂ SO₄. After removing the solvent 56 mg (97%) of a colourless oilwas obtained.

¹ H-NMR (CDCl₃): δ=0.8-0.95 (m; 6 H; 2×CH₂ --CH₃); 1.15-1.4 (m; 40 H;2×CO--(CH₂)₂ --(CH₂)₄, 2×(CH₂)₆ --CH₃); 1.5-1.9 (m; 12 H; 3×(--CH₂ --CH₂--CH₂ --)_(sperm), 2×CO--CH₂ --CH₂ --, (--CH--CH₂ --CH₂ --)_(sperm));1.9-2.1 (m, 8 H; 2×CH₂ --CH═CH--CH₂); 2.2-2.35 (m; 4 H; 2×CO--CH₂);2.45-3.3 (m; 10 H; 5×N--CH₂); 4.0-4.59 (m; 12 H; 2×CH--N, (CH(CH₂ O₂C--R)₂, 2×NH₂, 2×NH_(sperm)); 5.25-5.45 (m; 4 H; 2×CH═CH); 7.6-7.9(s(broad); 1 H; NH--CO).

¹³ C-NMR (CDCl₃): δ=14.0 (2×C(18)H₃); 22.5 (2×C(17)H₂); 24.7 (2×C(3)H₂);27.0, 27.1 (C(8)H₂, C(11)H₂); 29.0, 29.1, 29.2, 29.4, 29.60, 29.61, 31.8((CH--CH₂ --CH₂ --CH₂ --)_(sperm), 2×N--CH₂ --CH₂ --CH₂ --N, 2×C(4)H₂--C(7)H₂, 2×C(12)H₂ --C(16)H₂); 33.9 (2×C(2)H₂); 41.0, 47.3, 48.8(2×N--CH₂ --CH₂ --CH₂ --N, (CH--CH₂ --CH₂ --CH₂ --)_(sperm), (CH(CH₂ O₂C--R)₂); 62.0, 62.9 (CO--CH, CH(CH₂ O₂ C--R)₂); 129.5, 129.9 (2×CH═CH);173.4, 174.2 (CO--NH, 2×CH₂ --CCOO).

EXAMPLE 13 Synthesis of 2-dimethylamino-1,3-propanediol 13

456 mg (5 mmol) 2-amino-1,3-propanediol was placed in a round-bottomflask and admixed with 1.13 ml (25 mmol) formic acid (85%, d=1.20) whilecooling on ice. Subsequently 895 μl (12 mmol) formaldehyde (37%) wasadded and the mixture was heated for 10 h to 80° C. in a water bath.After cooling 3 ml 2 N hydrochloric acid was added. The product wasisolated by chromatography over 25 g acidic ion exchanger (DOWEX). Thecrude product obtained in this manner was purified by short-pathdistillation in an oil pump vacuum. 253 mg (42%) of a colourless oil wasobtained.

Boiling point_(1mbar) =105° C.

¹ H-NMR (DMSO-d₆): δ=2.25 (s; 6 H; N(CH₃)₂); 2.31-2.45 (m, 1 H; CH(CH₂OH)₂); 3.35-3.55 (m, 4 H; CH(CH₂ OH)₂); 4.24 (s(broad); 2 H; CH(CH₂OH)₂).

¹³ C-NMR (DMSO-d₆): δ=41.8 (N(CH₃)₂); 58.7 (CH(CH₂ OH)₂); 66.7 (CH(CH₂OH)₂).

EXAMPLE 14 Synthesis of 2-dimethylamino-(1,3-dioleoyloxy)-propane 14

A solution of 183 mg (1.54 mmol) 13, 1.088 g (3.85 mmol) oleic acid, 795mg (3.85 mmol) DCC and 18 mg (154 μmol) dimethylaminopyridine in 20 mlabs. CH₂ Cl₂ was stirred overnight at room temperature. Subsequently itwas filtered, rewashed with hexane/ether (3:1) and the filtrate wasrotary evaporated. After column chromatography on silica gel, (CH₂ Cl₂/MeOH=30:1) 444 mg (45%) of the product was obtained as a colourlessoil.

¹ H-NMR (CDCl₃): δ=0.85-0.95 (m; 6 H; 2×CH₂ --CH₃); 1.2-1.4 (m; 40 H;2×CO--(CH₂)₂ --(CH₂)₄, 2×(CH₂)₆ --CH₃); 1.55-1.7 (m; 4 H; 2×CO--CH₂--CH₂ --); 1.95-2.05 (m; 8 H; 2×CH₂ --CH═CH--CH₂); 2.3-2.35 (m, 4 H;2×CO--CH₂ --CH₂ --); 2.39 (s; 6 H; N(CH₃)₂); 2.9-3.0 (m; 1 H; CH(CH₂ O₂C--R)₂); 4.1-4.3 (m; 4 H; CH(CH₂ O₂ C--R)₂); 5.3-5.4 (m; 4 H; 2×CH═CH).

¹³ C-NMR (CDCl₃): δ=14.4 (2×C(18)H₃); 23.0 (2×C(17)H₂); 25.3 (2×C(3)H₂);27.55, 27.6 (C(8)H₂ ; C(11)H₂); 29.50, 29.53, 29.67, 29.7, 29.8, 29.9,30.06, 30.14, 32.3 (2×C(4)H₂ --C(7)H₂, 2×C(12)H₂ --C(16)H₂); 34.7(2×C(2)H₂); 42.4 (N(CH₃)₂); 61.6, 61.9 (CH(CH₂ O₂ C--R)₂); 130.09,130.36 (2×CH═CH); 173.9 (CH₂ --COO).

EXAMPLE 15 Synthesis ofN-[2-(1,3-dioleoyloxy)propyl]-N,N,N-trimethylammoniummethyl sulfate 15

66 μl (696 μmol) dimethyl sulfate was added at 0° C. to a solution of200 mg (309 μmol) 14 in 1.55 ml ethyl acetate/hexane (1:1) and thereaction mixture was stirred for 24 h at 4° C. Subsequently the solventwas removed. 213 mg (89%) of a colourless oil was obtained.

¹ H-NMR (CDCl₃): δ=0.75-0.9 (m; 6 H; 2×CH₂ --CH₃); 1.05-1.4 (m; 40 H;2×CO--CH₂)₂ --(CH₂)₄, 2×(CH₂)₆ --CH₃); 1.4-1.65 (m; 4 H; 2×CO--CH₂ --CH₂--); 1.8-2.1 (m; 8 H; 2×CH₂ --CH═CH--CH₂); 2.25-2.4 (m, 4 H; 2×CO--CH₂--CH₂ --); 3.3-3.45 (s(broad); 9 H; N(CH₃)₃); 3.61 (s; 3 H; CH₃ --O--S);4.1-4.2 (m; 1 H; CH(CH₂ O₂ C--R)₂); 4.4-4.65 (m; 4 H; CH(CH₂ O₂ C--R)₂);5.2-5.4 (m; 4 H; 2×CH═CH).

¹³ C-NMR (CDCl₃): δ=13.8 (2×C(18)H₃); 22.4 (2×C(17)H₂); 24.4 (2×C(3)H₂);26.9, 27.0 (C(8)H₂ ; C(11)H₂); 28.8, 28.9, 29.0, 29.05, 29.3, 29.46,29.5, 31.6 (2×C(4)H₂ --C(7)H₂, 2×C(12)H₂ --C(16)H₂); 33.6 (2×C(2)H₂);53.0, 54.0 (N(CH₃)₃, CH₃ --O--S); 58.7 (CH(CH₂ O₂ C--R)₂); 69.8 (CH(CH₂O₂ C--R)₂); 129.4, 129.8 (2×CH═CH); 172.3 (CH₂ --COO).

EXAMPLE 16N,N',N",N'"-tetra-tert.-butyloxycarbonyl-6-carboxyspermyl-2,3-dihydroxy-1-propylamide16

A solution of 300 mg (0.47 mmol) tetra-Boc-carboxy-spermine in 4 mldichloromethane was successively admixed with 59 mg (0.51 mmol)hydroxysuccinimide dissolved in 2 ml dichloromethane/THF (1:1) and 106mg (0.51 mmol) dicyclohexyl-carbodiimide dissolved in 2.2 mldichloromethane and stirred for 4 days at room temperature. Afterremoving the solvent the residue was taken up in ethyl acetate, filteredand the solvent was removed from the clear filtrate. The residue (335 mg(0.45 mmol) tetra-Boc-carboxyspermine-hydroxysuccinimidyl ester) wastaken up in 2.18 ml dimethylformamide and admixed with 41 mg (0.45 mmol)(±)-1-amino-2,3-propanediol and 61.5 μl (0.45 mmol) triethylamine. Afterstirring for 3 days the solvent was removed and the residue waspartitioned between ether and water. The organic phase is dried. Afterremoving the solvent an oily product was obtained which can be processeddirectly.

EXAMPLE 17 Synthesis ofN,N',N",N'"-tetra-tert.-butyloxycarbonyl-6-carboxy-spermyl-2,3-dioleoyloxy-1-propylamide17

218 mg (0.77 mmol) oleic acid, 180 mg (0.87 mmol)dicyclohexylcarbodiimide and a catalytic amount of dimethylaminopyridinewas added to a solution of 252 mg (0.35 mmol) 16 in 1.4 ml carbontetrachloride. It was stirred overnight. After removing the solvent theresidue was chromatographed (silica gel, dichloromethane with anincreasing content of methanol up to 10%). 392 mg (90% of theory) of anoil was obtained.

¹³ C-NMR: δ=173.8, 173.3 (oleic acid-C═O), 156 (C(═O)--N, 130.0,129.7(oleic acid C═C), 80.97, 79.67, 79.84 (quart.-BOC), 70.3 (Glyc. sn2),62.6 (Glyc. sn1), 59.1 (sperm.-), 46.5, 44.3, 43.7, 43.1 (sperm.N--CH₂), 39.5 (Glyc. sn3), 38.2 (sperm. N--CH₂), 34.9 (oleic acid C-2),31.9 (oleic acid C-16), 29.8-29.1 (oleic acid-CH₂), 28.4 (BOC-CH₃), 27.2(oleic acid C-8,11), 25.0 (oleic acid C-3), 22.7 (oleic acid C-17), 14.1(oleic acid C-18).

EXAMPLE 18 (6-Carboxy-spermyl)-2,3-dioleoyloxy-1-propylamide 18

A solution of 195 mg (0.16 mmol) 17 in 5 ml dichloromethane was admixedat room temperature with 5 ml TFA. After 20 min stirring it was dilutedwith 100 ml dichloromethane and washed with saturated sodium hydrogencarbonate solution. The combined organic phases were dried and thesolvent was evaporated. 134 mg (99%) of a viscous oil was obtained.

¹ H-NNR (500 MHz, CDCl₃ /CD₃ COOD 5:1) δ=5.33 (oleic acid 9, 10-H), 5.16(glycerol 2-H), 4.25 and 4.06 (glycerol 1-H), 4.15 (spermine-H), 3.55and 3.33 (glycerol 3-H), 3.18 and 3.05 (spermine N--CH₂), 2.31 (oleicacid 2-H), 2.21 (spermine --CH₂), 2.01 (oleic acid 8, 11-H), 1.98(spermine-), 1.83 (spermine CH₂), 1.57 (oleic acid 3-H), 1.30 (oleicacid 4-7, 12-17-H), 0.87 ppm (oleic acid 18-H).

¹³ C-NMR: δ=173.82 and 173.53 (oleic acid C-1), 154.06 (C(═O)--N),130.17 and 129.81 (oleic acid C-9 and C-10), 70 (glycerol C-2), 63.2(glycerol C-1), 39.8 (glycerol C-3), 34.25, 34.05, 33.97 (oleic acidC-2), 32.70, 31.93 (oleic acid C-16), 30.82, 29.79, 29.57 and 29.35(oleic acid CH₂), 27.25 (oleic acid C-8, 11), 25.64, 25.53, 25.32, 24.97(oleic acid C-3), 24.71, 22.70 (oleic acid C-17), 14.13 ppm (oleic acidC-18). MS (FAB): MH⁺ : 848.6 (calc. 848.8).

EXAMPLE 19 Synthesis of N-((N-Boc)-glycyl)-aminopropane-2,3-diol 19

10.00 g (36.73 mmol) Boc-Gly-Osu (Bachem) was dissolved together with3.35 g (36.77 mmol) 1-amino-2,3-propanediol in 50 ml DMF and stirred for18 h. The product was purified by flash chromatography (CH₂ Cl₂ /MeOH;95:5 v/v). 8.94 g (98%) 19 was obtained as a colourless oil.

¹ H-NMR (CDCl₃): δ=1.45 (s; 9 H; CH₃); 3.28 (m; 1 H; CH₂ --OH;); 3.39(m; 1 H; CH₂ --OH;); 3.35 (m; 1 H; CH₂ --N_(aminoprop).); 3.38 (m; 1 H;CH₂ --N_(aminoprop).); 3.78 (s; 3 H; CH--OH; CH₂ --NH_(gly)); 4.41 (brs; 1 H; OH); 4.67 (br s; 1 H; OH); 6.04 (tr; 1 H; NH); 7.45 (tr; 1 H;NH).

¹³ C-NMR (CDCl₃): δ=28.4 (CH₃); 42.2 (CH₂ --N_(gly)); 44.0 (CH₂--N_(aminoprop).); 64.0 (CH₂ --OH); 70.8 (CH--OH); 80.3 (quart.C_(Boc)); 156.6 (NCO₂); 174.5 (NCO).

EXAMPLE 20 Synthesis of 2,3-dioleoyloxy-N-((N-Boc)-glycyl)-aminopropane20

8.90 g (35.85 mmol) 19 was dissolved together with 22.70 g (110.02 mmol)DCC and 26.60 g (94.17 mmol) oleic acid in 50 ml DMF. 0.42 g (3.44 mmol)DMAP was added and stirred for 2 d. Afterwards it was filtered, rotaryevaporated and the product was purified by chromatography (CH₂ Cl₂/MeOH; 99:1 v/v). Yield 19.25 g 20 as a colourless oil (69%).

¹ H-NMR (CDCl₃): δ=0.89 (tr; 6 H; CH₃, oleic acid); 1.30 (br s; 40 H;C--CH₂ --C); 1.45 (br s; 9 H; CH₃, _(Boc)); 1.62 (m; 4 H; CH₂ --CH₂C═O); 2.03 (m; 8 H; CH₂ --CH═CH); 2.32 (tr; 4 H; CH₂ C═O); 3.42-3.57 (m;2 H; CH₂ --N_(aminoprop).); 3.75 (d; 2 H; CH₂ --N_(gly)); 4.12 (dd; 1 H;CH₂ --O_(aminoprop).); 4.27 (dd; 1 H; CH₂ --O_(aminoprop).); 5.08 (m; 1H; CH--O); 5.20 (s, 1 H; NH); 5.34 (m; 4 H; CH═CH); 6.58 (tr; 1 H; NH).

¹³ C-NMR (CDCl₃): δ=14.0 (CH₃, oleic acid); 22.7 (CH₂, oleic acid); 24-9(CH₂, oleic acid); 27.2 (CH₂, oleic acid); 27.2 (CH₂, oleic acid); 28.3(CH₂, oleic acid); 29.2 (CH₂, oleic acid); 29.3 (CH₃, Boc); 29.5 (CH₂,oleic acid); 29.8 (CH₂, oleic acid); 31.9 (CH₂, oleic acid); 34.0 (CH₂,oleic acid); 34.2 (CH₂, oleic acid); 39.7 (CH₂ --N_(gly)); 44.5 (CH₂--N_(aminoprop).); 62.7 (CH₂ --O_(aminoprop).); 70.2(CH--O_(aminoprop).); 80.3 (quart. C_(Boc)); 129.7 (CH═CH); 129.7(CH═CH); 130.0 (CH═CH); 130.0 (CH═CH); 156.1 (NCO₂); 169.8 (NCO); 173.2(CO_(oleic) acid); 173.4 (CO_(oleic) acid).

EXAMPLE 21 Synthesis of 2,3-dioleoyloxy-N-(glycyl)-aminopropane 21

15.20 g (19.56 mmol) 20 was taken up in 200 ml CH₂ Cl₂ /TFA (3:1 v/v)and stirred for 30 min. Afterwards the solution was diluted with 200 mlCH₂ Cl₂ and shaken out with 200 ml sat. NaHCO₃ solution. The organicphase was dried over MgSO₄ and rotary evaporated. Yield: 11.64 g (88%)21 as an oil.

¹ H-NMR (CDCl₃): δ=0.88 (tr; 6 H; CH₃); 1.29 (br s; 40 H; C--CH₂ --C);1.62 (m; 4 H; CH₂ --CH₂ C═O); 1.82 (s; 2 H CH₂ --N_(gly)); 2.01 (m; 8 H;CH₂ --CH═CH); 2.32 (tr; tr; 4 H; CH₂ C═O); 3.36 (s; 2 H; NH₂); 3.43-3.62(m; 2 H; CH₂ --N_(aminoprop).); 4.12 (dd; 1 H; CH₂ --O_(aminoprop).);4.28 (dd; 1 H; CH₂ --O_(aminoprop).); 5.14 (m; 1 H; CH--O_(aminoprop).);5.34 (m; 4 H; CH═CH); 7.60 (tr; 1 H; NH).

¹³ C-NMR (CDCl₃): δ=14.0 (CH₃); 22.6 (CH₂, oleic acid); 24.8 (CH₂, oleicacid); 24.8 (CH₂, oleic acid); 27.1 (CH₂, oleic acid); 29.0 (CH₂, oleicacid); 29.1 (CH₂, oleic acid); 29.2 (CH₂, oleic acid); 29.4 (CH₂, oleicacid); 29.6 (CH₂, oleic acid); 31.8 (CH₂, oleic acid); 34.0 (CH₂, oleicacid); 34.2 (CH₂, oleic acid); 39.1 (CH₂ --N_(gly)); 44.5 (CH₂--N_(aminoprop).); 62.7 (CH₂ --N_(aminoprop).); 129.6 (CH_(oleic) acid);129.9 (CH_(oleic) acid); 173.1 (NCO); 173.1 (CO_(oleic) acid); 173.3(CO_(oleic) acid).

EXAMPLE 22 Synthesis of2,3-dioleoyloxy-N-(N-(N,N',N",N'"-tetra-tert.-butyloxycarbonyl-6-carboxy-spermyl)-glycyl)-aminopropane22

100 mg (0.15 mmol) 21 was dissolved together with 102 mg (0.16 mmol)(Boc)₄ -spermyl-COOH and 39 mg (0.19 mmol) DCC in 1 ml CH₂ Cl₂ andstirred for 18 h. Afterwards precipitated DCH was removed by filtration.Concentration by evaporation and chromatographic purification (CH₂ Cl₂/MeOH, 97:3) yielded 123.1 mg (63%) 22 as a colourless oil.

¹ H-NNR (CDCl₃ +0.1 ml CD₃ COOD): δ=0.88 (tr; 6 H; CH₃, oleic acid);1.28 (br s; 40 H; C--CH₂ --C_(oleic) acid); 1.46 (br s; CH₃,Boc ;CH₂,spermyl); 1.62 (m; 4 H; CH₂ CH₂ C═O); 2.03 (m; 8 H; CH₂ --CH═CH);2.32 (m; 4 H; CH₂ C═O); 3.00-3.33 (m; 10 H; CH₂, spermyl); 3.33-3.62 (m;2 H; CH₂ --N_(aminoprop).); 3.95 (m; 2 H; CH₂ --N_(gly)); 4.10 (m; 1 H;CH₂ --O); 4.28 (m; 1 H; CH₂ --O); 4.30 (br s; 1 H; CH_(spermyl)); 5.12(m; 1 H; CH_(aminoprop).); 5.35 (m; 4 H; CH═CH).

¹³ C-NMR (CDCl₃ +0.1 ml CD₃ COOD): δ=14.1 (CH₃, oleic acid); 22.8 (CH₂,oleic acid); 25-0 (CH₂, oleic acid); 27.3 (CH₂, oleic acid); 27-3 (CH₂,oleic acid); 28.4 (CH₃, Boc); 28.5 (CH₃, Boc); 29.3 (CH₂, oleic acid);29.3 (CH₂, oleic acid); 29.4 (CH₂, oleic acid); 29.7 (CH₂, oleic acid);29.9 (CH₂, oleic acid); 32.0 (CH₂, oleic acid); 34.2 (CH₂, oleic acid);39.7 (CH₂ --N_(gly)); 43.1 (CH₂ --N_(aminoprop).); 63.0 (CH₂ --O); 70.2(CH--O); 79.6 (quart. C_(Boc)); 80.2 (quart. C_(Boc)); 80.9 (quart.C_(Boc)); 81.5 (quart. C_(Boc)); 129.8 (CH═CH); 130.1 (CH═CH); 155-158(NCO₂); 173.6 (COO); 173.8 (COO).

EXAMPLE 23 Synthesis of2,3-dioleoyloxy-N-(N-(6-carboxy-spermyl)-glycyl)-aminopropane 23

1.00 g (0.77 mmol) 22 was dissolved in 100 ml CH₂ Cl₂ /TFA (3:1) andstirred for 30 min. Subsequently the solvent was removed. 1.03 g(98.80%) 23 was obtained as a wax-like product.

¹ H-NMR (d₆ -DXSO): δ=0.85 (tr; 6 H; CH₃,oleic acid); 1.23 (br s; 44 H;C--CH₂ --C_(oleic) acid/spermyl); 1.50 (m; 4 H; CH₂ --CH₂ C═O); 1.67 (m;2 H; C--CH₂ --C_(spermyl)); 1.83 (m; 2 H; C--CH₂ --C_(spermyl)); 1.95(m; 8 H; CH₂ --CH═CH); 2.23 (m; 4 H; CH₂ C═O); 2.82-3.30 (m; 10 H; CH₂,spermyl); 3.26 (m; 1 H; CH₂ --N_(aminoprop).); 3.36 (m; 1 H; CH₂--N_(aminoprop).); 3.74-3.88 (m; 2 H; CH₂ --N_(gly)); 3.93 (br s; 1 H;CH_(spermyl)); 4.01 (m; 1 H; CH₂ --O); 4.24 (m; 1 H; CH₂ --O); 5.02 (m;1 H; CH_(aminoprop).); 5.31 (m; 4 H; CH═CH); 8.10 (br s; 6 H; NH); 8.33(tr; 1 H; NHC═O); 8.98 (br s; 2 H; NH); 9.04 (tr; 1 H; NHC═O); 9.15 (brs; 1 H; NH); 9.50 (br s; 1 H; NH).

¹³ C-NMR (d₆ -DMSO): δ=13.8 (CH₃,oleic acid); 20.9 (CH₂, sper); 22.1(CH₂, oleic acid); 23.8; 23.9 (CH₂, sper); 24.4, 26.6 (CH₂, oleic acid);26.9 (CH₂, sper); 28.5-29.1 (CH₂, oleic acid); 31.3 (CH₂, oleic acid);33.4, 33.6 (CH₂,oleic acid); 36.2 (CH₂, sper); 38.7 (CH₂, sper); 41.8(CH₂ --N_(gly)); 43.0 (CH₂ --N_(aminoprop).); 43.9, 46.2 (CH₂, sper);58.8 (CH_(sper)); 62.8 (CH₂ --O); 129.5 (CH═CH); 129.6 (CH═CH); 167.4,168.5 (NCO_(sper/gly)); 172.2 (COO); 172.4 (COO).

EXAMPLE 24 Transfection of Adherent HELA Cells with CAT Plasmid

1. Starting Material

1. Medium for the HELA stock culture (ATTC No. CCL 2): MEM (with EARL'ssalts), 10% FCS, 2 mM pyruvate, 2 mM glutamine, 1×n.e. amino acids.

For the transfection prepare the same medium containing 5% FCS and thesame medium without FCS.

2. pSV2-CAT plasmid in TE buffer, concentration: 1 mg/ml, 5 kb. (GormanC. M. et al., Mol. Cell. Biol. 2, 1044-1051(1982)).

3. New transfection reagents:

in MES (20 mM MES, 150 mM NaCl, pH 6.2) concentration 1.0 mg/ml,sonicated (Branson sonifier) and sterilized by filtration or

in 99.8% EtOH, concentration 2.5 mg/ml (not sonicated and not sterilizedby filtration)

4. HEPES: 20 mM Hepes, 150 mM NaCl, pH 7.4, sterile

5. PBS, BM order No. 210 331 (10 mM buffer, 150 mM salt), sterile andunsterile

6. 150 mM NaCl,

7. lysis buffer: 10 mM MOPS, NaCl 10 mM, 1 mM EGTA, 1% mM Triton X-100,pH 6.5.

2. Transfection Mixture

One day before transfection the cells were transferred to 6 cm petridishes: for this the cells were trypsinized and diluted to 2×10⁵cells/ml (cell count determined with a Neubauer counting chamber) inmedium containing 5% FCS. 5 ml per dish. Incubation in an incubator (at37° C., 5% CO₂).

Mixture Per Dish for Aqueous Solutions of the Transfection Reagent (TR):

1. Add HEPES to 5 μg plasmid (=5 μl) to a final volume of 100 μl andvortex.

2. Add HEPES to 10-40 μg TR (=10-40 μl) to a final volume of 100 μl andvortex.

3. Combine the solutions from 1. and 2., shake carefully. Allow to standfor 10 to 15 min at RT.

4. During the incubation period change the medium in the test dishes:aspirate the old medium and replace with 3 ml medium (containing 5% FCSor, if without FCS, wash cells twice with PBS).

5. Add plasmid-TR mixture from 3 (200 μl) directly to the fresh medium(with or without FCS) and disperse evenly by carefully swirling thedish.

6. Incubate for 6 h in an incubator (at 37° C., 5% CO₂).

7. Then supplement the medium to a final concentration of FCS of 5-10%.Final medium volume 6 ml.

8. Incubate for 19 h in an incubator.

9. Then completely aspirate the mixture and replace with 5 ml mediumcontaining 5-10% FCS.

Mixture Per Dish for Ethanolic Solutions of the TR:

A Without FCS in the Medium:

1.5 μg plasmid (=5 μl)+500 μl medium without FCS, and vortex.

2. 10-40 μg TR (=4-16 μl)+500 μl medium without FCS, and vortex.

3. Wash cells free of FCS: aspirate 5 ml medium and wash twice with PBS;aspirate.

4. Combine solutions from 1. and 2., shake carefully.

5. Place plasmid-TR mixture from 4. (ca. 1020 μl) onto the washed cellsand distribute equally by carefully swirling the dish.

6. Incubate for 6 h in an incubator (at 37%, 5% CO₂).

7. Then supplement the medium to a final concentration of FCS of 5-10%.Final medium volume 6 ml.

8. Incubate for 18 h in an incubator.

9. Then completely aspirate the mixture and replace by 5 ml mediumcontaining 5-10% FCS.

B With FCS in the Medium

1. 5 μg plasmid (=5 μl)+150 μl NaCl and vortex.

2. 10-40 μg TR (=4-16 μl)+150 μl NaCl and vortex.

3. Combine solutions from 1 and 2, briefly shake carefully. Allow tostand for 15 min at RT.

4. During the incubation period change the medium in the test dishes:aspirate the old medium and replace with 1.5 ml medium (containing 10%FCS).

5. Place plasmid-TR mixture from 3 (ca. 320 μl) onto the fresh mediumand distribute equally by carefully swirling the dish.

6. Incubate for 6 h in an incubator (at 37%, 5% CO₂).

7. Then supplement the medium to a final concentration of FCS of 5-10%.Final medium volume 6 ml.

8. Incubate for 18 h in an incubator.

9. Then completely aspirate the mixture and replace by 5 ml mediumcontaining 5-10% FCS.

3. Cell Lysis

1. Aspirate the medium and wash the cells three times with ice-cold PBS.

2. Carefully aspirate residual liquid and add 1 ml lysis buffer perdish. Incubate for 30 min at RT, allow the dishes to stand withoutshaking.

3. Remove the cell lysate by pipette and transfer to 1.5 ml Eppendorftubes.

4. Centrifuge the lysates in a bench centrifuge, 3 min.

5. Remove the supernatant and transfer into a fresh vessel. Discard thepellet.

4. Protein Determination of the Lysates

The protein determination is carried out after the lysis.

The protein determination is analysed according to Bradford (M.Bradford, Anal. Biochem. 72, 248 (1976)).

For the further examination the lysates from an experiment are adjustedwith sample buffer (from the CAT-ELISA-kit, Boehringer Mannheim, orderNo. 1363727) to the same protein concentration (ca. 250 μg/ml).

5. Determination of the Transfection Efficiency

The lysates are examined in a CAT-ELISA (Boehringer Mannheim, order No.1363727). 200 μl of each of the lysates adjusted to the same proteinconcentration is used in the ELISA.

Comparison of the absorbances yields information about the transfectionefficiency.

Reconstitution of the Kit Components and Dilution to WorkingConcentration:

1. CAT Standard, Bottle 1

Add 0.5 ml redistilled water to a bottle of lyophilisate and dissolve byshaking. The concentration (ng/ml) is printed on the label.

Working Dilution:

Dilute the dissolved lyophilisate (cf. bottle label for theconcentration) with sample diluent buffer (bottle 7) to 1.0, 0.5, 0.25,0.125, 0.0625, 0.0312, 0 ng/ml.

2. PAB<CAT> Dig, Bottle Z

Add 0.5 ml redistilled water to a bottle of lyophilisate and dissolve byshaking (concentration=0.2 mg/ml).

Working Dilution:

Dilute the dissolved lyophilisate (0.2 mg/ml) with sample diluent buffer(bottle 7) to 2 μg/ml.

3. PAB<Dig> POD, Bottle 3

Add 0.5 ml redistilled water to a bottle of lyophilisate and dissolve byshaking (concentration=20 U/ml).

Working Dilution:

Dilute the dissolved lyophilisate (20 U/ml) with sample diluent buffer(bottle 7) to 150 mU/ml.

4. Washing Buffer, Bottle 6

Ready-to-use washing buffer is prepared by mixing 1 part 10× washingbuffer (bottle 6) with 9 parts redistilled water. 1× washing buffer isrequired for all washing steps.

5. Sample Diluent Buffer, Bottle 7

Bring bottle no. 7 to RT in a water bath. Ready to use.

6. POD Substrate, Bottle 4

ABTS 1-component substrate (bottle No. 4), ready to use, temperaturekept constant at RT.

Procedure

The following table refers to the buffers and working dilutionsmentioned above. The MTP modules are tapped out on multiple cellulosepaper layers after each washing step.

For the 37° C. incubation steps an MTP cover foil is stuck onto theplate.

    __________________________________________________________________________    Working step     Volume                                                                              Time Temp.                                                                              shaking                                      __________________________________________________________________________      remove MTP module strip        -                                              2. CAT enzyme standard, working 200 μl/well 60 min 37° C. -                                          dilutions or cell lysates                    3. discard, wash 3 times with 300 μl/well  18-25° C. -                                               washing buffer, tap out                      4. PAB<CAT>DIG 200 μl/well 60 min 37° C. -                           working dilution                                                             5. discard, wash 3 times with 300 μl/well  18-25° C. -                                               washing buffer, tap out                      6. PAB<DIG>POD 200 μl/well 60 min 37° C. -                           working solution                                                             7. discard, wash 3 times with 300 μl/well  18-25° C. -                                               washing buffer, tap out                      8. substrate solution 200 μl/well 10 min 18-25° C. +                9. measure at 405 nm after ca.                                                 10 min substrate incubation                                                __________________________________________________________________________

The results are shown in FIGS. 1 and 2.

We claim:
 1. A compound of the formula (I) ##STR2## in which R₁ is asaturated or unsaturated C(O)--C₁₋₂₃ or saturated or unsaturated C₁₋₂₄chain,A is selected from A₁ and A₂ A₁ is an O--R₂ group in which R₂ hasthe meaning stated for R₁ and can be the same as or different from R₁,A₂ is an NR₃ X or an N⁺ R₃ R₄ R₅ Y⁻ residue in whichR₃, R₄ areindependently selected from the group consisting of hydrogen, an alkylgroup with 1 to 4 C atoms, a (CH₂)_(n) --OH and a (CH₂)_(n) --NH₂ groupwhere n=2-6, provided that R₃ and R₄ are not both hydrogen, R₅ which canbe the same as or different from R₃ and R₄, is hydrogen, an alkyl groupwith 1 to 4 C atoms, a (CH₂)_(n) --OH, a (CH₂)_(n) -halogenide or a((CH₂)_(m) NH)_(o) --(CH)₂)_(n) group in which m is an integer from 2 to6 and is equal to or different from n and o wherein n is a number from 2to 6 and o is an integer from 0 to 4, X has the meaning for R₅ or hasthe following meaning: an amidically bound amino acid, an amidicallybound peptide or polypeptide, a C(O)--CHR₆ N(R₇)₂, a C(O)--CHR₆ N⁺(R₇)₃, a C(O)--CHR₆ N⁺ (R₇)₂ R₈ or a C(O)--CHR₆ NR₇ R₈ group, providedthat if R₃ X is an amidically bound amino acid, an amino acidderivative, a peptide or polypeptide, wherein,R₆ is a (CH₂)_(m) --NR₇R₈, a (CH₂)_(m) --N⁺ (R₇)₃ R₈ or a (CH₂)_(m) --N⁺ (R₇)₂ R₈ residue and mis a number from 1 to 5, R₇ is hydrogen or an alkyl group with 1 or 4 Catoms, R₈ is a (CH₂)_(n) --N(R₇)₂ or (CH₂)_(n) --N⁺ (R₇)₃ group in whichn is a number from 2 to 4 and R₇ can have the meaning stated above and Yis a pharmaceutically acceptable anion, B is selected from B₁ and B₂, B₁is an NH[C(O)--(CH₂)_(p) --NH]_(q) --Z residue in which p is a numberfrom 1 to 6 and q is a number from 0 to 2,Z is an amidically bound aminoacid, an amidically bound peptide or polypeptide, a C(O)--CHR₆ N(R₇)₂, aC(O)--CHR₆ N⁺ (R₇)₃, a C(O)--CHR₆ N⁺ (R₇)₂ R₈ or a C(O)--CHR₆ NR₇ R₈group wherein R₆ to R₈ and m have the aforementioned meanings and B₂ hasthe meaning stated for A₁ and when A is A₁, B is B₁ and when A is A₂, Bis B₂.
 2. A compound as claimed in claim 1, wherein the residue R₁represents a saturated or unsaturated C₁₀₋₂₀ or C(O)₁₀₋₂₀ group.
 3. Acompound as claimed in claim 1, wherein A₂ denotes NR₃ X in which R₃ ishydrogen and X is an amidically bound amino acid, an amino acidderivative, a peptide or polypeptide.
 4. A compound of the generalformula (I) ##STR3## in which R₁ represents a saturated or unsaturatedC(O)--C₁₋₂₃ or saturated or unsaturated C₁₋₂₄ chain,A₁ represents anO--R₂ group in which R₂ has the meaning stated for R₁ and can be thesame as or different to R₁ A₂ represents an NR₃ X or an N⁺ R₃ R₄ R₅ Y⁻residue in whichR₃, R₄ which can be the same as or different to oneanother represent hydrogen, an alkyl group with 1 to 4 C atoms, a(CH₂)_(n) --OH or a (CH₂)_(n) --NH₂ group where n=2-6, R₅ which can bethe same as or different to R₃ or R₄, denotes hydrogen, an alkyl groupwith 1 to 4 C atoms, a (CH₂)_(n) --OH, a (CH₂)_(n) -halogenide, or a((CH₂)_(m) NH)_(o) --(CH₂)_(n) --NH₂ group in which m is an integer from2 to 6 and can be equal or different to n or o wherein n can be a numberfrom 2 to 6 and o an integer from 0 to 4, X can, in addition to themeaning for R₅, have the following meaning: an amidically bound aminoacid, an amidically bound peptide or polypeptide, a C(O)--CHR₆ N(R₇)₂, aC(O)--CHR₆ N⁺ (R₇)₃, a C(O)--CHR₆ N⁺ (R₇)₂ R₈ or a C(O)--CHR₆ NR₇ R₈group in whichR₆ can be a (CH₂)_(m) --NR₇ R₈, a (CH₂)_(m) --N⁺ (R₇)₃ ora (CH₂)_(m) --N⁺ (R₇)₂ R₈ residue and m can be a number from 1 to 5, R₇represents hydrogen or an alkyl group with 1 to 4 C atoms, R₈ representsa (CH₂)_(n) --N(R₇)₂ or (CH₂)_(n) --N⁺ (R₇)₃ group in which n is anumber of 2 to 4 and R₇ can have the meaning stated above and Y is apharmaceutically acceptable anion, B₁ represents a NH[C(O)--(CH₂)_(p)--NH]_(q) --Z residue in which p is a number from 1 to 5 and q is 0 or 1and Z represents a C(O)--CHR₆ N(R7)₂, a C(O)--CHR₆ N⁺ (R7)₃, aC(O)--CHR₆ N⁺ (R7)₂ R₈ group and R₆ to R₈ and m have the aforementionedmeanings B₂ can have the meaning stated for A₁ and the meaning for A₁ isonly valid with B₁ and that of A₂ is only valid with B₂.
 5. A compoundas claimed in claim 1, wherein Y denotes a halogenide, monomethylsulfate, acetate, trifluoroacetate or phosphate.
 6. A compound asclaimed in claim 1, wherein said compound is selected from the groupconsisting of 2-(6-carboxyspermyl)-1,3-dioleoyloxy-propylamide,1-(6-carboxyspermyl)-2,3-dioleoyloxy-propylamide,2,3-dioleoyloxy-N-(N-(6-carboxyspermyl)-glycyl)-aminopropane,2-(6-carboxyspermyl)-1,3-dimyristoyloxy-propylamide,2-(1,1,1,5,5,10,10,14,14,14-deca-methyl-6-carboxyspermyl)-1,3-dioleoyloxy-propylamide2-(N,N,N,N',N',N'-hexamethylornithyl)-1,3-dioleoyloxy-propylamide and2-(N,N,N,N',N',N'-hexamethyllysyl)-1,3-dioleoyloxy-propylamide.
 7. Areagent comprising of at least one compound of the general formula (I),and contains a water-miscible solvent and formula (I) is defined asfollows: ##STR4## in which R₁ represents a saturated or unsaturatedC(O)--C₁₋₂₃ or saturated or unsaturated C₁₋₂₄ chain,A₁ represents anO--R₂ group in which R₂ has the meaning stated for R₁ and can be thesame as or different to R₁ A₂ represents an NR₃ X or an N⁺ R₃ R₄ R₅ Y⁻residue in whichR₃, R₄ which can be the same as or different to oneanother represent hydrogen an alkyl group with 1 to 4 C atoms, a(CH₂)_(n) --OH or a (CH₂)_(n) --NH₂ group where n=2-6, R₅ which can bethe same as or different to R₃ or R₄, denotes hydrogen, an alkyl groupwith 1 to 4 C atoms, a (CH₂)_(n) --OH, a (CH₂)_(n) -halogenide or a((CH₂)_(m) NH)_(o) --(CH₂)_(n) --NH₂ group in which m is an integer from2 to 6 and can be equal or different to n or o wherein n can be a numberfrom 2 to 6 and o an integer from 0 to 4, X can, in addition to themeaning for R₅, have the following meaning: an amidically bound aminoacid, an amidically bound peptide or polypeptide, a C(O)--CHR₆ N(R₇)₂, aC(O)--CHR₆ N⁺ (R₇)₃, a C(O)--CHR₆ N⁺ (R₇)₂ R₈ or a C(O)--CHR₆ NR₇ R₈group whereinR₆ can be a (CH₂)_(m) --NR₇ R₈, a (CH₂)_(m) --N⁺ (R₇)₃ or a(CH₂)_(m) --N⁺ (R₇)₂ R₈ residue and m can be a number from 1 to 5, R₇represents hydrogen or an alkyl group with 1 to 4 C atoms, R₈ representsa (CH₂)_(n) --N(R₇)₂ or (CH₂)_(n) --N⁺ (R₇)₃ group in which n is anumber from 2 to 4 and R₇ can have the meaning stated above and Y is apharmaceutically acceptable anion, B₁ represents an NH[C(O)--(CH₂)_(p)--NH]_(q) --Z residue in which p is a number from 1 to 6 and q is anumber from 0 to 2,Z represents an amidically bound amino acid, anamidically bound peptide or polypeptide, a C(O)--CHR₆ N(R₇)₂, aC(O)--CHR₆ N⁺ (R₇)₃, a C(O)--CHR₆ N⁺ (R₇)₂ R₈ or a C(O)--CHR₆ NR₇ R₈group and R₆ to R₈ and m have the aforementioned meanings and B₂ canhave the meaning stated for A₁ and the meaning for A₁ is only valid withB₁ and that of A₂ is only valid with B₂.
 8. A reagent as claimed inclaim 7, further comprising at least one lipidic compound in at leastone water-miscible solvent.
 9. A reagent as claimed in claim 7, whereinthe water-miscible solvent is a lower alcohol.
 10. A reagent comprisingat least one compound as claimed in claim 1 in the form of liposomes orother aggregates.
 11. A method for the delivery of molecules into cells,comprising contacting a reagent of claim 7 with an anionic biomoleculeand subsequently with cells for transfection.
 12. A method as claimed inclaim 11, wherein the molecules comprise at least one of DNA and RNA.13. A method as claimed in claim 11, wherein the anionic biomolecule isDNA or a corresponding fragment.
 14. A method as claimed in claim 11,wherein said at least one compound in said reagent is selected from thegroup consisting of 2-(6-carboxyspermyl)-1,3-dioleoyloxy-propylamide,1-(6-carboxyspermyl)-2,3-dioleoyloxy-propylamide,2,3-dioleoyloxy-N-(N-(6-carboxyspermyl)-glycyl)-aminopropane,2-(6-carboxyspermyl)-1,3-dimyristoyloxy-propylamide,2-(1,1,1,5,5,10,10,14,14,14-deca-methyl-6-carboxyspermyl)-1,3-dioleoyloxy-propylamide2-(N,N,N,N',N',N'-hexamethylornithyl)-1,3-dioleoyloxy-propylamide and2-(N,N,N,N',N',N'-hexamethyllysyl)-1,3-dioleoyloxy-propylamide.
 15. Areagent as recited in claim 10, further comprising at least one otherlipidic compound.
 16. A method as recited in claim 14, wherein saidreagent further comprises at least one other lipidic compound.